RESEARCH ARTICLE
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Development 136, 563-574 (2009) doi:10.1242/dev.016816
The cell migration molecule UNC-53/NAV2 is linked to the
ARP2/3 complex by ABI-1
Kristopher L. Schmidt1,2, Nancy Marcus-Gueret1,2, Adetayo Adeleye1,2, Jordan Webber1, David Baillie2 and
Eve G. Stringham1,2,*
The shape changes that are required to position a cell to migrate or grow out in a particular direction involve a coordinated
reorganization of the actin cytoskeleton. Although it is known that the ARP2/3 complex nucleates actin filament assembly, exactly
how the information from guidance cues is integrated to elicit ARP2/3-mediated remodeling during outgrowth remains vague.
Previous studies have shown that C. elegans UNC-53 and its vertebrate homolog NAV (Neuronal Navigators) are required for the
migration of cells and neuronal processes. We have identified ABI-1 as a novel molecular partner of UNC-53/NAV2 and have found
that a restricted calponin homology (CH) domain of UNC-53 is sufficient to bind ABI-1. ABI-1 and UNC-53 have an overlapping
expression pattern, and display similar cell migration phenotypes in the excretory cell, and in mechanosensory and motoneurons.
Migration defects were also observed after RNAi of proteins known to function with abi-1 in actin dynamics, including nck-1, wve-1
and arx-2. We propose that UNC-53/NAV2, through its CH domain, acts as a scaffold that links ABI-1 to the ARP2/3 complex to
regulate actin cytoskeleton remodeling.
INTRODUCTION
The successful development of multicellular organisms requires that
cells and cellular processes migrate sometimes long distances to
well-defined target locations. To achieve such a journey, the cell
must repeatedly reorganize its cytoskeleton in response to a
multitude of navigational cues. The nematode Caenorhabditis
elegans has proven to be an excellent model organism for the
elucidation of global guidance mechanisms controlling migration.
Several signals and receptors reported to control cell migration in
vertebrates have been described in C. elegans, including: UNC6/netrin and its receptors UNC-40/DCC and UNC-5 (Hedgecock et
al., 1990; Ishii et al., 1992), the SLT-1/SLIT cue (Hao et al., 2001)
and its multifunctional receptor SAX-3/ROBO (Ghenea et al., 2005;
Levy-Strumpf and Culotti, 2007; Zallen et al., 1998), WNTs and
their associated FZ receptors (Pan et al., 2006), and growth factors
such as EGL-17/FGF (Burdine et al., 1997) and its receptor EGL15/FGFR (Birnbaum et al., 2005; Bulow et al., 2004; DeVore et al.,
1995). Besides the identification of the signaling cues and their
receptors, the machinery required for the subcellular positioning of
receptors is also being uncovered. For example, recent evidence
suggests that the kinesin-like molecule VAB-8 localizes SAX-3/
ROBO to the cell surface to modulate the response to SLT-1
guidance cues (Watari-Goshima et al., 2007), and that both MIG-2/
RhoGTPase and VAB-8 are required for the localization of the
UNC-40 receptor (Levy-Strumpf and Culotti, 2007).
At the leading edge of motile cells, local reorganization of the
actin cytoskeleton is mediated by the ARP2/3 complex, which
nucleates the assembly of branched actin filaments (Higgs and
Pollard, 2000; Volkmann et al., 2001). Potential regulators of
ARP2/3 include the ABI proteins, initially discovered to function as
1
Department of Biology, Trinity Western University, 7600 Glover Road, Langley,
BC V2Y 1Y1, Canada. 2Department of Molecular Biology and Biochemistry, 8888
University Drive, Simon Fraser University, Burnaby, BC V5A 1S6, Canada.
*Author for correspondence (e-mail: stringha@twu.ca)
Accepted 9 December 2008
downstream targets of ABL non-receptor tyrosine kinases
implicated in RAC-dependent cytoskeletal organization and
remodeling (Courtney et al., 2000; Funato et al., 2004; Jenei et al.,
2005; Stradal et al., 2001), in addition to processes such as cell
migration, neuronal development, growth cone pathfinding and
endocytosis (Courtney et al., 2000; Grove et al., 2004; Ibarra et al.,
2005; So et al., 2000). ABI family members localize to the actin-rich
tips of lamellipodia and filopodia, and are widely expressed in the
developing murine nervous system (Courtney et al., 2000; Stradal
et al., 2001). ABI forms part of the pentameric WAVE complex
together with NAP-1, PIR121/SRA-1, HSPC300 and WAVE, which
has been shown to mediate actin remodeling through ARP2/3
(Bompard and Caron, 2004; Takenawa and Suetsugu, 2007). The
WAVE complex members WVE-1, GEX-2 (SRA-1) and GEX-3
(NAP-1) have been characterized in C. elegans. GEX-2 and GEX3 colocalize to cell boundaries during embryogenesis and interact
physically (Soto et al., 2002), and loss of WVE-1 or the GEXs
results in defective hypodermal cell migration, incomplete ventral
enclosure during morphogenesis, and embryonic lethality (Soto et
al., 2002; Withee et al., 2004). Additionally, ventral enclosure
defects in WVE-1 and GEX mutants are reminiscent of those found
in RAC and ARP2/3 mutant animals, suggesting that a conserved
pathway involving RAC, the WAVE complex and ARP2/3 is
maintained during embryogenesis in C. elegans (Sawa and
Takenawa, 2006; Soto et al., 2002; Withee et al., 2004).
While much has been uncovered with respect to the signaling
events at the cell surface and the mechanics of actin filament
assembly, relatively little is known about the proteins that integrate
guidance information to instruct cytoskeletal reorganization. One
candidate is UNC-53, initially discovered to control the migration
of a subset of cells and cellular extensions along the anterior to
posterior axis in C. elegans. Hypomorphic alleles of unc-53 display
reduced extension and guidance defects in the outgrowth of the
mechanosensory neurons (Hekimi and Kershaw, 1993), the
excretory canals (Hedgecock et al., 1990; Stringham et al., 2002)
and the sex muscles, the latter resulting in an egg-laying defective
phenotype in hermaphrodites (Stringham et al., 2002). By contrast,
DEVELOPMENT
KEY WORDS: Cell migration, Axonal guidance, Cytoskeleton, ARP2/3 complex, C. elegans
RESEARCH ARTICLE
overexpression of UNC-53 in muscle cells results in exaggerated
outgrowth during embryogenesis (Stringham et al., 2002). UNC-53
interacts genetically and physically with the SH2-SH3 adaptor
protein SEM-5 (GRB-2), a mediator of EGL-15/FGFR signaling in
sex myoblast migration in C. elegans (Chen et al., 1997; Stringham
et al., 2002), suggesting a role for UNC-53 in signal transduction.
UNC-53 also contains several domains observed in actin-binding
proteins, suggesting a possible function in actin cytoskeleton
dynamics (Stringham et al., 2002).
Roles in cell migration have also been documented for the
vertebrate homologs of UNC-53. Three human UNC-53 homologs
(NAV1, NAV2 and NAV3; Neuron Navigator 1, 2 and 3) have been
identified (Maes et al., 2002; Merrill et al., 2002). The most similar
homolog to UNC-53, NAV2, is retinoic acid inducible in the
developing nervous system (Merrill et al., 2002), and hypomorphic
mice have sensory deficits subsequent to morphological defects that
are consistent with a role for NAV2 in neuron outgrowth (Peeters et
al., 2004). NAV1 associates with microtubule plus-ends on
developing neuronal growth cones and is required for netrin-induced
directionality in pontine neurons (Martinez-Lopez et al., 2005). The
NAV proteins are also expressed in a range of adult tissues,
including brain, heart and kidney, in both mice and humans
following outgrowth (Maes et al., 2002; Martinez-Lopez et al.,
2005; Peeters et al., 2004), and Nav3 mRNA is localized to synapses
at neuromuscular junctions (Kishi et al., 2005).
In this study, we show that the largest UNC-53 isoforms are
expressed in several migrating cells in which UNC-53 is required,
as well as in adult cells. Additionally, we show that UNC-53 binds
the C. elegans ABI-1 homolog, and that loss of ABI-1 leads to
migration defects similar to those found when unc-53, wve-1, nck-1
and arx-2 (arp2) are removed. We find that UNC-53 and ABI-1 have
an overlapping pattern of expression, and that disruption of the
interaction between UNC-53 and ABI-1 impairs longitudinal
guidance. Finally, we propose a model for how UNC-53 might
function with ABI-1 and the ARP2/3 complex in cytoskeletal
remodeling.
MATERIALS AND METHODS
C. elegans strains
C. elegans Bristol (N2) and mutant strains were maintained according to
standard protocols (Brenner, 1974). Strains used in this study include:
BC06288 ppgp12::gfp (sIs10089);
ZB171 pmec-4::gfp (bzIs7);
BC12924 pnck-1::gfp (sIs12798 [dpy-5(e907)/dpy-5(e907); sEx12728
[rCesZK470.5::GFP + pCeh361]);
BC14371 punc-53L::gfp (sEx14371 [dpy-5(e907)/dpy-5(e907);
sEx14371 [rCesF45E10.1a::GFP + pCeh361]);
BC10129 pabi-1::gfp (sEx10129 [dpy-5(e907)/dpy-5(e907); sEx14371
[rCesB0336.6::GFP + pCeh361]);
VA97 pabi-1::abi-1::gfp (pmEx97 [pabi-1::abi-1(1-1410)::GFP +
pRF4]);
VA71 unc-53(n166); sIs10089;
VA106 unc-53(n152); sIs10089;
VA72 unc-53(n166); bzIs7;
FX494 abi-1(tm494) (684bp deletion III: 5690340…5691023);
VA74 abi-1(tm494); sIs10089;
VA99 abl-1(ok171); sIs10089;
VA100 wsp-1(gm324); sIs10089;
VA75 abi-1(tm494); bzIs7;
VA76 nck-1(ok694); sIs10089 [nck-1(ok694) contains a 1814bp deletion
X: 4150378…4152191];
VA77 nck-1(ok694); bzIs7;
VA79 unc-53(n166); abi-1(tm494); sIs10089;
Development 136 (4)
VA91-VA92 abi-1(tm494); sIs10089; pmEx91-92 [ppgp12::abi-1(11410) + pDPY-30::NLS::DSRED2 (Cordes et al., 2006)];
VA93-VA96 sIs10089; pmEx93-96 [ppgp12::unc-53(1-416)::GFP +
pDPY-30::NLS::DSRED2 (Cordes et al., 2006)];
VA103-VA104 unc-53(n152); sIs10089; pmEx103-104 [ppgp12::unc53(1-4965) + pDPY-30::NLS::DSRED2 (Cordes et al., 2006)];
VH715 nre-1(hd20); lin-15b(hd126); hdIs17; hdIs10; and
VA78 eri-1(mg366); bzIs7.
Yeast two-hybrid assays
A NdeI-NcoI fragment of the unc-53 cDNA (nucleotides 64-480
corresponding to amino acids 1 to 139) was subcloned into pAS2
(Matchmaker, Clontech Laboratories) to generate pVA200. Using pVA200
as bait and a mixed stage C. elegans cDNA library as prey (gift of Bob
Barstead, Oklahoma Medical Research Foundation, Oklahoma, USA)
candidate binding partners were identified in a yeast two-hybrid screen by
assaying for growth on triple drop-out media (–Trp-Leu-His) and the βgalactosidase activity of doubly transformed yeast Y190 cells as described
(Aspenstrom and Olson, 1995). Six of the positives corresponded to the
B0336.6/abi-1 locus. pVA305 corresponds to the shortest abi-1 cDNA
isolated in the screen cloned into the pSE1107 vector, coding for amino acids
12 to 427 of ABI-1, and was used for subsequent biochemical studies.
In vitro binding assays
ABI-1::GST and UNC-53N::6⫻His protein were purified from BL21(DE3)
E. coli harboring pVA600 and pVA63, respectively. pVA600 was generated
by inserting a 1.3-kb XhoI fragment from pVA305 into pGEX4T2
(Invitrogen). Recombinant proteins ABI-1::GST or GST were expressed and
purified according to the manufacturer’s instructions (Invitrogen), with
modifications (Frangioni and Neel, 1993). Ten micrograms of ABI-1::GST
or GST were applied to glutathione resin for pull-down experiments. Total
UNC-53N::6⫻His lysates (25 μg) were applied to ABI-1::GST or GST
glutathione resin and incubated at 4°C for 3 hours. Following incubation,
beads were washed four times with 20 mM Tris pH 7.4, 0.1 mM EDTA, 300
mM NaCl and 0.1% Triton X-100, and bound proteins were extracted and
analyzed by SDS-PAGE and western blotting.
RNA interference and mutant analysis
RNAi experiments were performed by feeding (Kamath et al., 2001), using
RNAi clones obtained from Geneservice, with the exception of the unc-53L
RNAi clone pVA504, which was generated by cloning a 0.3-kb XhoI-NcoI
PCR fragment corresponding to nucleotides 1 to 280 (exons 1-4) of the unc53 cDNA from pTB113 (Stringham et al., 2002) in tandem into pPD129.36
(gift of Andrew Fire, Stanford University School of Medicine, Stanford,
USA). Animals carrying the ppgp-12::gfp reporter were scored for excretory
canal outgrowth with respect to the position of the gonad arms, the vulva and
the anus. Neuronal RNAi was carried out using either the neuronal enhanced
sensitive strain eri-1(mg366); pmec-4::gfp for mechanosensory neurons
(Kennedy et al., 2004), or nre-1(hd20); lin15b(hd126) for motoneurons. The
anterior process of the PLM neuron was scored as abnormal if the stop point
was posterior to the wild-type position at the mid-body. Ventral cord
motoneurons commissures were determined to have defects if two or more
axons exhibited ectopic lateral branching or stalling and were unable to
reach the dorsal cord.
Preparation of UNC-53 and ABI-1 polyclonal antisera
A SacI-NcoI fragment of the unc-53 cDNA (nucleotides 64-480) that
corresponds to amino acids 1 to 139 was subcloned into the expression
vector pRSET (Amersham Pharmacia) to generate pVA63, which was then
expressed in E. coli BL21 cells according to manufacturer’s protocols
(Invitrogen). Purified protein was emulsified in Titre Max Gold and injected
into a female New Zealand white rabbit. The antiserum collected was active
at titers of 1:30,000 on western blots of recombinant fusion protein. The
specificity of PAb-UNC-53N was confirmed by staining animals that
ectopically express UNC-53 in the intestine under control of the hsp-16
promoter (Stringham et al., 1992). Whereas no staining was observed in
control animals, strong staining in the cytoplasm of intestinal cells was
observed after heat shock. For the generation of ABI-1 polyclonal antibody
(PAb-ABI-1), a synthesized peptide (DYNSIYQPDRYGTIRAGGR)
DEVELOPMENT
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UNC-53/NAV-2 interacts with ABI-1
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containing amino acids 256-274 from ABI-1 was coupled to keyhole limpet
hemocyanin (KLH) and used to immunize guinea pigs (Open Biosystems).
Anti-sera were affinity purified towards the ABI-1 peptide and were active
at a dilution of 1:10,000 on western blots of recombinant protein.
Expression pattern of UNC-53L and ABI-1
To detect UNC-53L and ABI-1 in vivo by immunostaining, staged larvae or
adults were fixed and permeabilized according to the method of Ruvkun and
Finney (Bettinger et al., 1996). PAb-UNC-53N was used at 1:500 dilution
and PAb-ABI-1 antibody at 1:100, with secondary anti-Rabbit IgG
(Invitrogen Molecular Probes) and anti-Guinea Pig IgG (Open Biosystems)
used at 1:500 and 1:400, respectively. Anti-GFP immunostaining was
performed using chick anti-GFP primary at 1:100 with secondary rabbit antiChick IgG at 1:400 (Millipore, USA), directed towards the VA97 strain.
VA97 contains the extrachromosomal array pmEx97 generated by coinjecting 1 ng/μl of the pabi-1::abi-1::gfp, containing 0.5 kb upstream of
abi-1 and the entire abi-1 locus fused to GFP with the pRF4 co-injection
marker at 100 ng/μl. Expression of abi-1 and unc-53L were also determined
using promoter GFP transcriptional fusions. The pabi-1::gfp fusion
(BC10129) included 275 bp upstream of abi-1 fused to GFP, whereas 2.9 kb
upstream of unc-53 was fused to GFP to generate BC14371 (McKay et al.,
2003). Specimens were viewed with Olympus IX81 or Leica DMLB
fluorescence microscopes using appropriate filter sets.
Cell autonomy and overexpression experiments
Rescue of the abi-1(tm494) posterior canal defects was achieved by coinjecting 10 ng/μl of the PCR fusion ppgp12::abi-1, containing the pgp-12
promoter (Zhao et al., 2005) fused to full-length abi-1, with 100 ng/μl of the
DEVELOPMENT
Fig. 1. Characterization of the long isoforms (UNC-53L) of unc-53. (A) Structure of the unc-53 gene. The start of the various UNC-53L and
UNC-53S isoforms are indicated by arrows. The promoter for UNC-53SA is between exons 5 and 8, and the promoter for UNC-53SB is located
between exons 8 and 13 (Choi and Newman, 2006; Stringham et al., 2002). 2.9 kb of DNA upstream of the transcriptional start site of UNC-53LA
was used to construct punc-53L::gfp. Alternatively spliced exons are shown in pink. unc-53(n152) is a 319-bp deletion removing parts of exons 18
and 19, producing a stop codon in exon 20 (Stringham et al., 2002), and n166 is a single nucleotide C to T transition in exon 19 that introduces a
premature stop codon. (B) The longest polypeptide, UNC-53LA, is 1654 amino acids and contains a calponin homology domain (CH, red; amino
acids 11-109), two LKK motifs (LKK, purple; 114-133 and 1097-1116), two proline-rich SH3-binding motifs (SH3b, green; 487-495 and 537-545),
two coiled-coil regions (CC, blue; 890-923 and 1078-1113) and an AAA domain (yellow; 1292-1425). n166 introduces a premature stop codon at
amino acid 949. Both n152 and n166 remove the coiled-coil, LKK and AAA domains from all isoforms. The first five exons of UNC-53 (UNC-53N;
amino acids 1-139) were used for the production of PAB-UNC-53N antisera and the GAL4 DNA-binding domain (GAL4DBD) in pVA200 for yeast
two-hybrid studies. (C,D) Expression pattern using punc-53L::gfp. (C) Adult hermaphrodite (anterior is left), showing GFP expression in head (HNeu)
and tail (TNeu) neurons, the excretory cell (EXc), and the ventral nerve cord (VC). (D) Midbody, showing expression in the sex myoblasts (SM) and
the ventral cord (VC). (E,F) Expression pattern using PAb-UNC-53N antisera. (E) Expression of UNC-53L throughout the excretory cell and canals
(EXc). (F) UNC-53L expression in a pair of coelomocytes (CC). Scale bars: 100 μm.
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Development 136 (4)
Fig. 2. ABI-1 physically interacts with the
N terminus of UNC-53. (A) ABI-1::GAL4AD
interacts with UNC-53N::GAL4DBD by yeast
two-hybrid assay. Yeast harboring both bait
(pVA200) and prey (pVA305) plasmids grow
in triple drop-out media (–Trp-Leu-His) and
are positive for β-galactosidase, in contrast
to yeast transformed with empty vectors
(pAS2 and pSE1107), or with either the bait
or prey vector alone. (B) GST pull-down
assay. Equal amounts of ABI-1(12-427)-GST
and GST alone were expressed in E. coli and
bound to glutathione-conjugated beads
(Bound). Soluble UNC-53N::6His lysates (left
lane) were incubated with protein-bound
beads and were observed to bind ABI-1(12427)-GST (middle lane) but not GST alone
(right lane), as detected by PAb-UNC-53N.
RESULTS
Characterization of UNC-53L
The unc-53 locus is large, contains 23 exons, and encodes six
polypeptides, the largest consisting of 1654 amino acids (Fig. 1A,B)
(Stringham et al., 2002). Several protein motifs are shared between
UNC-53 and its orthologs, including a calponin homology (CH)
domain, two putative actin-binding sites of the LKK motif, two
coiled-coil regions, two polyproline-rich SH3-binding motifs, and an
AAA (ATPases associated with diverse cellular activities) domain
(Stringham et al., 2002). A combination of alternative trans-splicing
and cis-splicing events and the presence of two intronic promoters
gives rise to four large isoforms of UNC-53 (LA, LB, LC and LD),
collectively referred to as UNC-53L, and two small isoforms of UNC53 (SA and SB), referred to as UNC-53S. Previous studies indicate
that the intronic promoters that transcribe UNC-53SA and UNC-53SB
have complementary non-overlapping expression patterns (Stringham
et al., 2002). To characterize the expression of the UNC-53L isoforms,
a promoter region 2.9-kb upstream of the most 5⬘ SL1 splice site of
unc-53 was used, and strong GFP expression was observed in the
excretory cell, in neurons in head and tail ganglia, and in several
classes of ventral cord motoneuron (Fig. 1C), as well as in the sex
myoblasts (Fig. 1D). Immunostaining with an antibody raised against
the translated product of the first 5 exons of unc-53 (Fig. 1B) revealed
UNC-53L protein localized in the cytoplasm of the excretory cell (Fig.
1E), the sex myoblasts, the anal muscles, the head neurons and the
coelomocytes (Fig. 1F). Expression was detected at all developmental
stages, including adults.
UNC-53L interacts with Abelson Interactor-1 (ABI-1)
Previous studies suggest that UNC-53 functions in signal
transduction during migration (Stringham et al., 2002), yet few
molecules known to interact directly with UNC-53 have been
identified. Moreover, a phage cDNA devoid of the first four exons
was sufficient to partially rescue the Unc and Egl phenotypes of unc53 mutants (Stringham et al., 2002). Therefore, we wondered what
molecules might interact with the N-terminal of UNC-53, as this
region is conserved in NAV2 and NAV3. To answer this question,
we screened for interactors in a C. elegans yeast two-hybrid library,
using the N terminus (UNC-53N) as bait (Fig. 1B). Of the
candidates isolated, six corresponded to the B0336.6 genomic locus,
the C. elegans abi-1 (Abelson Interactor-1) homolog (Fig. 2A). Of
the abi-1 clones isolated, the smallest cDNA encoded amino acids
12-427, thereby excluding the SH3 region of ABI-1 as a required
domain for UNC-53 binding. Moreover, the UNC-53 bait contained
only the first 139 amino acids of UNC-53L, and thus was devoid of
the polyproline repeats that are typical of SH3-binding domains.
Therefore, the interaction between ABI-1 and UNC-53 does not
appear to be mediated by SH3 binding. The yeast two-hybrid data
was further confirmed by GST pull down (Fig. 2B), demonstrating
that the interaction between ABI-1 and UNC-53 is direct.
Characterization of abi-1
BLAST analysis revealed the presence of a single abi-1 gene in C.
elegans. The abi-1 genomic locus spans 2624 bp and contains five
exons encoding a predicted polypeptide of 469 amino acids (Fig. 3).
C. elegans ABI-1 is a conserved protein that is homologous to ABI1 of C. briggsae (CAE64316), Homo sapiens (NP_005461), Mus
musculus (Q3TJ64) and Drosophila melanogaster (NP_477263).
The highest degree of homology resides within several recognizable
protein domains suggestive of function. Near the N terminus is a QSNARE motif (Echarri et al., 2004), which in the case of
mammalian ABI-1, has been shown to be involved in growth factor
receptor endocytosis and to bind Syntaxin 1 (Echarri et al., 2004;
Tanos and Pendergast, 2007). Partially overlapping with the QSNARE motif is an ABL-homeodomain homologous region that is
similar to the DNA-binding region of homeodomain proteins, and
which is found exclusively in adaptor proteins that interact with Ablfamily tyrosine kinases (Dai and Pendergast, 1995). Additionally, C.
elegans ABI-1 contains a serine-rich region and multiple
polyproline-rich putative SH3-binding motifs. A single SH3 domain
is located at the C terminus of C. elegans ABI-1 and this domain has
been shown to mediate the interaction of ABI-1 with Abelson
tyrosine kinases (Shi et al., 1995), and to control the ability of ABL
to phosphorylate downstream targets, including mammalian enabled
(Mena) (Tani et al., 2003). The abi-1(tm494) allele (Mitani
Laboratory, National Bioresource Project) carries a 684-bp deletion
DEVELOPMENT
plasmid pDPY-30::NLS::DSRED2 (Cordes et al., 2006) into the strain VA74
to create vaEx91 and vaEx92. unc-53(n152) posterior canal defects were
rescued by co-injecting the PCR fusion ppgp12::unc-53L (100 ng) with
pDPY-30::NLS::DSRED2 into VA106 and scoring transgenic unc-53(n152)
homozygous animals as described. UNC-53CH-expressing arrays vaEx93vaEx96 were generated by co-injecting ppgp-12::unc-53CH::gfp containing
the pgp-12 promoter fused to the first 422 nucleotides of unc-53 cDNA from
pVA63 and GFP at 100 ng/μl, along with 100 ng/μl of the plasmid pDPY30::NLS::DSRED2 (Cordes et al., 2006) into the strain BC06288. Excretory
canal morphology was scored in young adult animals co-expressing GFP
and dsRED from all lines for general defects and for posterior canal
migration position, as described above.
UNC-53/NAV-2 interacts with ABI-1
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beginning at nucleotide 1659 in exon 4 (Fig. 3A), and encodes a
predicted truncated product ending at proline 350 followed by
TGPVRL and a premature stop codon, resulting in a 356 amino acid
product (Fig. 3C).
Overlap in unc-53 and abi-1 mutant phenotypes
UNC-53 controls the migration of several cells and cellular
processes along the anteroposterior axis in C. elegans, including the
sex myoblasts (Chen et al., 1997), the ALN/PLN and ALM/PLM
axons, and the excretory cell (Stringham et al., 2002). To determine
whether the observed interaction between ABI-1 and UNC-53 is
relevant in vivo, the migration phenotypes of abi-1 were
characterized. The migration and outgrowth of both the anterior and
posterior excretory canal processes were visualized using the
transgenic reporter ppgp-12::gfp, which is expressed exclusively in
the excretory cell from the 3-fold embryonic stage onwards (Zhao
et al., 2005). The excretory cell (EC) is a large H-shaped cell in C.
elegans and represents an excellent cell type for the study of both
dorsoventral and anteroposterior migrations. During its outgrowth,
two processes emerge from the EC body and migrate dorsolaterally
from the ventral side of the terminal pharyngeal bulb towards the
lateral hypodermis. In wild-type animals, when the canals reach the
lateral hypodermis they cross the hypodermal basement membrane
and bifurcate, sending processes anteriorly to the head and
posteriorly to the tail (Nelson et al., 1983) (Fig. 4A).
By contrast, although the excretory cell body is positioned
normally in unc-53(n166) animals, both the anterior and posterior
canals are severely truncated. In the case of the anterior processes,
they terminate close to the excretory cell body, often not extending
further than the anterior pharyngeal bulb, while the majority of
posterior canals grow out approximately half way, terminating at the
level of the vulva (Fig. 4B). These phenotypes were also observed
DEVELOPMENT
Fig. 3. Molecular organization of C. elegans ABI-1
gene and protein. (A) Gene structure of abi-1. The
boundaries of introns, exons and the abi-1(tm494)
deletion are indicated. (B) Protein structure of ABI-1.
ABI-1 contains a Q-SNARE domain (Q-SNARE, green;
amino acids 56-110) (Echarri et al., 2004), an Ablinteractor homeodomain homologous region (ABLHHR, purple; 95-173), a serine-rich region (Ser-rich,
orange; 243-259), three proline-rich SH3-binding motifs
(SH3b, red; 296-306, 347-351 and 371-374) and an
SH3 domain (SH3, blue; 416-469). ABL-HHR and SH3
domains were predicted using the Simple Modular
Architecture Research Tool (SMART, http://smart.emblheidelberg.de). (C) Comparison of C. elegans ABI-1 with
C. briggsae, human, mouse and Drosophila orthologs.
Multiple alignments were performed using Clustal W
1.83 (http://align.genome.jp) with ABI-1 proteins from
C. elegans (CE29545), C. briggsae (CAE64316), H.
sapiens (NP_005461), M. musculus (Q3TJ64) and D.
melanogaster (NP_477263), and were drawn using
BOXSHADE (http://www.ch.embnet.org). The peptide
sequence used to generate ABI-1 antibody PAb-ABI-1 is
indicated in blue and the start site of the tm494
deletion is indicated by an asterisk.
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Development 136 (4)
by unc-53(rnai) and in unc-53(n152) mutants (Fig. 4C,H). Notably,
defects in the posterior migrations of the excretory canals in unc53(n166) animals were equally as strong as in heterozygous
n166/mnDf87 worms (Fig. 4H), suggesting that unc-53(n166),
which removes a coiled-coil domain, an LKK motif and the AAA
cassette from all isoforms (Fig. 1B), is a null allele for this
phenotype. To determine the role of abi-1 in excretory cell
migration, we examined the length of canals in abi-1(tm494) and by
RNAi. Animals carrying the abi-1(tm494) deletion are superficially
wild type, with the exception of a mild uncoordination defect,
characterized by an increased frequency of backing. Although the
cell body of the excretory cell in abi-1(tm494) is positioned
normally, examination of the posterior excretory canals of tm494
revealed a failure of the majority to exit the gonad region (Fig.
4E,H). The position of the excretory cell body was also normal in
abi-1(rnai) animals, but a range of migration defects were observed,
including truncated anterior and posterior excretory canals (Fig.
4C,H), which is reminiscent of unc-53 loss-of-function alleles, in
addition to dorsoventral defects not observed in unc-53 mutants
(data not shown) and excretory cell cysts (Fig. 4G). Notably,
excretory canal defects were observed to be no more severe in unc53(n166); abi-1(tm494) double mutants than in unc-53(n166) (Fig.
4H). Although the deletion in abi-1(tm494) results in a truncated
protein, the abi-1(tm494) allele behaves as a hypomorph, as abi1(rnai) exacerbates excretory canal defects in an abi-1(tm494)
background (data not shown). As is the case for the abi-1(tm494);
DEVELOPMENT
Fig. 4. Excretory canal morphology in wildtype, unc-53 and abi-1 animals.
(A-G) Fluorescence micrographs of
hermaphrodites carrying the ppgp-12::gfp
transgene, allowing for the visualization of the
excretory cell body and canals (anterior and
posterior termini marked by arrows, A-E).
Anterior is to the left and animals are displayed
laterally with the exception of B and E, which
are shown ventrally. (A) Morphology of the wildtype excretory cell body and processes. The
excretory cell body is positioned on the ventral
side of the posterior pharyngeal bulb and
extends two canals towards the anterior of the
animal to the tip of the head and two canals
posteriorly to the tail. (B) unc-53(n166). (C) unc53(rnai). (D) abi-1(rnai). (E) abi-1(tm494).
(F,G) Lateral view of wild-type excretory canal (F)
and abi-1(rnai) canal (G), showing numerous
small cysts (arrows). (H) Quantification of
posterior excretory canal outgrowth defects.
The outgrowth of the posterior canals was
divided into three regions (1-3) between the
vulva and the tail as shown. The stop point of
canals was determined by fluorescence
microscopy for wild type (n=72), abi-1(rnai)
(n=141), abi-1(tm494) (n=116), unc-53(rnai)
(n=87), unc-53(n152) (n=37), unc-53(n166)
(n=55), unc-53(n166)/mnDf87 (n=107),
unc-53(n166); abi-1(rnai) (n=55), unc-53(n166);
abi-1(tm494) (n=55), abi-1(tm494);
ppgp-12::abi-1 (n=51) and unc-53(n152);
ppgp-12::unc-53L (n=28). Scale bars: 100 μm.
UNC-53/NAV-2 interacts with ABI-1
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569
Fig. 5. Mechanosensory neuron phenotype in wild-type, unc-53 and abi-1 animals. (A) Fluorescence micrograph showing a lateral view of a
wild-type hermaphrodite carrying the pmec-4::gfp transgene. The PLM, PVM and ALM neuronal cell bodies and axons are shown. The stop point of
the PLM axon was scored with respect to the wild-type position near the ALM cell body (arrowheads indicate final position of the PLM axon).
(B) unc-53(n166) animal with truncated PLM and PVM axons. (C) unc-53(rnai) animal with truncated PLM and PVM axons. (D) abi-1(rnai) animal
showing truncated PLM axon stopping short of the PVM cell body. (E) AVM axon in a wild-type animal. AVM axons were considered wild type if
they projected anteriorly past the nerve ring (NR) and terminated at the tip of the animal. (F) abi-1(tm494) animal showing an AVM axon
misdirected dorsally (arrow), followed by an abnormal posteriorly directed migration. Scale bars: 100 μm.
observed (Fig. 5E,F). During wild-type development, the AVM axon
undergoes a short ventral migration followed by a turn and migration
anteriorly along the ventral cord, where it extends past the nerve ring
into the tip of the head (Fig. 5E). In abi-1(tm494) animals, the initial
ventral and longitudinal anterior migrations of the AVM axons were
wild type but, instead of terminating along their anterior trajectory,
the AVM would occasionally reroute, turning dorsally away from
the ventral cord and then posteriorly back towards the nerve ring
(Fig. 5F).
ABI-1 and UNC-53 have overlapping expression
patterns
Given the physical interaction between UNC-53 and ABI-1, the
shared phenotypes, and because UNC-53 has been shown to
function cell autonomously (Stringham et al., 2002), we expected
that ABI-1 would be found in the same cells as UNC-53. To
determine the expression pattern of ABI-1, we generated
transcriptional and translational GFP-reporter fusions of abi-1 and
raised a polyclonal antibody towards a peptide corresponding to
amino acids 256 to 274 of ABI-1. Collectively, these approaches
revealed that ABI-1 is expressed in a number of neurons within the
nerve ring and head, including the amphid interneurons AIYL/R
(Fig. 6A), the RMEL/R motoneurons (Fig. 6B), coelomocytes (Fig.
6D), and several classes of ventral cord motoneuron, where it is
localized throughout the cell bodies, dendrites and commissural
axons extending to the dorsal cord (Fig. 6C), similar to the pattern
of expression observed for UNC-53L transcripts (Fig. 1C).
ABI-1 and UNC-53 control dorsoventral migrations
of the motoneurons
The expression of both UNC-53 and ABI-1 in motoneurons suggests
that both genes may have a role in the guidance and outgrowth of
these cells. Consistent with this, RNAi of abi-1 revealed multiple
DEVELOPMENT
unc-53(n166) double mutant, abi-1(rnai) in an unc-53(n166)
background does not reduce the extension of the posterior excretory
canals beyond that of unc-53(n166) alone (Fig. 4H), suggesting that
these genes function within the same pathway to control outgrowth
of the posterior excretory canals. Previous work suggests that unc53 may function cell autonomously (Stringham et al., 2002), so
given the physical interaction observed between UNC-53 and ABI1, we hypothesized that ABI-1 and UNC-53 may both function
within the excretory canals. Consistent with this prediction, fulllength abi-1 and unc-53 cDNA driven by the ppgp-12 excretory cellspecific promoter was sufficient to rescue the canal outgrowth
defects of abi-1(tm494) and unc-53(n152) mutants, respectively
(Fig. 4H).
To determine whether ABI-1 functions in the migration of axons,
we examined the outgrowth of the mechanosensory neurons that
control the touch response. The cell bodies and axons of the
mechanosensory neurons are reproducibly positioned in wild-type
animals, and can be visualized using the cell-specific pmec-4::gfp
reporter. In wild-type animals, PLML/R cell bodies are positioned
within the lumbar ganglia of the tail, where they extend a short
posteriorly directed process and a long anteriorly directed axon that
grows out along the lateral commissure and terminates near the cell
bodies of the ALM neurons (Fig. 5A). By contrast, in unc-53(n166)
animals, both the anterior and posterior processes are shorter than in
wild type. The anterior axons of the PLML/R were prematurely
truncated and terminate shortly after beginning their trajectory (Fig.
5B). Similar phenotypes were observed in unc-53(rnai) (Fig. 5C).
Although no posterior defects were observed in abi-1(rnai) animals,
a significant number of anteriorly directed PLM axons terminated
prematurely at positions posterior to the ALML/R cell bodies (Fig.
5D). None of the PLM axons was shorter in abi-1(rnai) animals than
in unc-53(n166). In the weak abi-1(tm494) allele, outgrowth of PLM
was normal but misdirection of the AVM axonal projection was
570
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Development 136 (4)
defects in motoneurons, including the presence of ectopic branches
in commissures, giving rise to disorganized neural networks (Fig.
6F,G). Frequently, dorsally directed axons were unable to complete
their migration to the dorsal cord (32%, n=90) and either bifurcated
prematurely, extending lateral processes anteriorly and posteriorly,
or produced several knob-like structures in disoriented processes,
which was suggestive of growth cone stalling (Fig. 6F).
Defasciculation of the ventral cord was also frequently observed
(13%, n=90; Fig. 6H). Similar phenotypes have been reported in
unc-53, where approximately 13% of motoneuron commissures are
abnormal and fail to reach the dorsal cord (Stringham et al., 2002).
Disruption of UNC-53L causes defects in cell
outgrowth
Although the N terminus of UNC-53 inclusive of a CH domain is
sufficient to bind ABI-1 in vitro, this region is absent in short UNC53 isoforms (Stringham et al., 2002). To determine whether the longisoform of UNC-53 is required for posterior EC migration, we
directed RNAi toward exons 1-4 of UNC-53 and found that
knockdown of UNC-53L is sufficient to impair longitudinal
guidance even with the short isoforms present (Fig. 7A), a finding
that is consistent with UNC-53L function and expression in the EC.
To test further whether the interaction between UNC-53 and ABI-1
is functionally important, we overexpressed the CH domain of
UNC-53L in the excretory cell. At a low frequency, various canal
defects were observed, including ectopic outgrowths, cysts and the
truncation of posterior canals (Fig. 7B-D), phenotypes that are
reminiscent of abi-1 knockdown. This suggests that expression of
the CH domain alone may act in a dominant-negative fashion to
sequester endogenous ABI-1 and prevent functional interaction with
wild-type UNC-53 in vivo.
Mutations in actin-polymerization proteins
disrupt longitudinal migration
The extension of cellular processes is mediated primarily through the
extension of growth cones, highly motile ends at cell tips that are
undergoing constant cytoskeletal reorganization. ABI-1 functions in
cytoskeletal organization through its ability to regulate the ARP2/3
complex to induce actin polymerization. Evidence suggests that ABI1 may regulate ARP2/3 through a complex with WAVE in response
to RAC (Bompard and Caron, 2004; Stradal et al., 2004) or by binding
WASP (Innocenti et al., 2002). To test whether these and other proteins
known to function with ABI-1 are involved in longitudinal migration
in C. elegans, we analyzed the excretory canal phenotypes of mutant
and RNAi-treated animals. Of the genes tested, wve-1(rnai), nck1(ok694) and arx-2(rnai) produced excretory canal migration
phenotypes reminiscent of unc-53 and abi-1 mutants (Fig. 8), while
wsp-1 and abl-1 did not. Notably, none of the genes tested had more
severe phenotypes either alone or in the background of the null unc53 allele (n166), suggesting that the initial trajectory of the posterior
excretory canals to the anterior gonad arm is unaffected by loss of unc53, abi-1 or known abi-1 interactors. Interestingly, nck-1 is expressed
in the excretory cell and ventral cord motoneurons (Fig. 9), two cell
types affected in unc-53 and abi-1 mutant backgrounds. We also
examined the potential role of these proteins in the migration of PLM
axons and found that RNAi had modest effects (Table 1).
DISCUSSION
A role for ABI-1 in cell migration and growth cone
extension
In this study, ABI-1 was localized to the cytoplasm of ventral cord
motoneurons, as well as to commissures that span the dorsoventral
axis and innervate the dorsal cord. The expression pattern is
DEVELOPMENT
Fig. 6. ABI-1 expression and motoneuron migration defects. (A-D) Expression pattern of ABI-1. (A) Head of a larva expressing pabi-1::gfp in
the AIYL/R neurons. (B,C) Expression of ABI-1 detected by immunostaining with PAb-ABI-1. (B) Head of a wild-type animal showing ABI-1 expression
in the nerve ring and in the cell bodies of the RMEL/R neurons (arrow). (C) Ventral surface of the midbody of an adult worm showing ABI-1 in the
cell bodies of motoneurons (arrowheads), in addition to the longitudinal tracts of the ventral cord (VC) and the dorsal commissures (arrow).
(D) Expression of ABI-1 in coelomocyte (CC). (E-H) Fluorescence micrographs showing wild-type (E) and abi-1(rnai) (F-H) animals. Ventral is down in
all cases except H. (E) Wild-type animal with motoneuron cell bodies located ventrally and dorsal commissures extending to the dorsal cord. (F) abi1(rnai) animal showing a truncated and misguided dorsal commissure (arrowhead), and a truncated dorsal commissure with anterior and posterior
ectopic lateral branches (arrow). (G) Misrouting and branching of dorsal commissures (arrows). (H) Ventral view of the ventral cord of an abi-1(rnai)
animal showing marked defasciculation (arrows). Scale bars: 50 μm.
UNC-53/NAV-2 interacts with ABI-1
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571
Fig. 7. RNAi of the long isoform of UNC-53 and overexpression of
the UNC-53 CH domain generates excretory cell defects. (A) RNAi
directed toward the long isoform of unc-53 results in posterior canal
migration, with posterior excretory canals terminating at the vulva
(arrow). Three percent of animals had canals terminating at the vulva
and 24% failed to exit past the posterior gonad arm (n=92). (B) Adult
animal expressing ppgp12::unc-53CH::gfp in the excretory cell body, as
indicated by merge (yellow, arrowhead) with dsRED co-injection marker
pDPY-30::NLS::DSRED2, displays a posterior excretory canal truncated
at the midbody position near the vulva (arrow). Seven percent of canals
terminate near the vulva and 22% failed to exit the posterior gonad
arm (n=116). (C,D) Defects of the excretory canals in adult animals
expressing ppgp-12::unc-53CH::gfp in the excretory canal. (C) Head of
an adult animal showing cysts (arrow) and ectopic anterior branches
(arrowhead). (D) Adult animal showing various excretory canal cysts
(arrows). Scale bars: 100 μm.
consistent with a role for ABI-1 in nervous system development, and
is confirmed by the guidance defects observed in the motoneurons
of abi-1 animals. In mammals, ABI proteins are highly expressed in
the developing brain, where they guide nerve cell placement and
axon outgrowth (Courtney et al., 2000; Grove et al., 2004). ABI-1
localizes to the motile tips of lamellipodia and filopodia, which is
consistent with a role for ABI-1 in actin-polymerization events
(Echarri et al., 2004; Stradal et al., 2001). Surprisingly, ABI-1
expression was not seen in the excretory cell as had been expected
given the physical interaction between UNC-53 and ABI-1, and
because UNC-53 is expressed in the EC (Stringham et al., 2002). It
is possible that endogenous levels of ABI-1 are very low and/or that
sequences required for expression in these cells were absent in the
reporter fusions. Nonetheless, the ability of both ABI-1 and UNC-
ABI-1 interactors and ARP2/3 control longitudinal
migration
The migration defects observed in both abi-1 and unc-53 mutants is
consistent with the biochemical interaction observed between them.
Moreover, the phenotype of the ABI-1 interactor nck-1 in the
excretory cell suggests a similar role for nck-1. The adaptor NCK-1
exerts its influence in part through the modulation of WVE-1, as
NCK-1 and/or RAC activation is able to release SCAR-1 from an
inhibitory complex containing ABI-2 to activate the ARP2/3
complex (Eden et al., 2002). Therefore, it was not surprising that
similar longitudinal guidance phenotypes were also observed in
wve-1 and arx-2, suggesting that the primary mode of unc-53 action
in these migrations is mediated through conserved interactions.
Consistent with this view, both UNC-53 and the RAC activator
UNC-73/TRIO have been implicated in an EGL-17/FGFindependent signaling mechanism controlling sex myoblast
migration (Chen et al., 1997), suggesting that modulation of the
ARP2/3 complex may be the crucial determinant of actin filament
assembly in this migration as well. Interestingly, the first part of the
posteriorly directed migration of the excretory canals to the anterior
gonad arm was intact for all genes tested, suggesting that another
mechanism independent of unc-53, abi-1 and the ARP2/3 complex
might be driving the initial posterior outgrowth of the canals.
Experiments in both cell culture and model systems reveal that cell
shape changes, and the extension of cellular processes are mediated
through GTPases of the RHO family, including CDC42 and RAC,
which induce the formation of lamellipodia and filopodia by
interacting directly or indirectly with the WASP family of proteins,
resulting in the activation of the ARP2/3 complex and directed actin
nucleation (Bompard and Caron, 2004; Stradal et al., 2004; Takenawa
and Suetsugu, 2007). For example, in C. elegans, loss of WSP-1 or
WVE-1 disrupts hypodermal cell migration and ventral enclosure
during embryogenesis, phenotypes that are also characteristic of CED10/RAC-1 mutants (Lundquist et al., 2001) and ARP2/3-complex
knockdown (Sawa et al., 2003). Moreover, the motoneuron guidance
DEVELOPMENT
53 to rescue outgrowth when expressed specifically in the excretory
cell, coupled with the finding that disruption of the UNC-53–ABI1 interaction interfered with canal outgrowth, strongly suggest that
ABI-1 and UNC-53 function together in the excretory cell.
The expression of both ABI-1 and UNC-53 in motoneurons, and
the observation that loss of abi-1 and unc-53 function disrupts the
dorsal outgrowth of motoneuron commissures suggests that these
genes may participate together in dorsoventral guidance decisions. A
role for the navigators, vertebrate homologs of UNC-53, in dorsal
ventral guidance is also apparent. Mouse NAV1 is expressed in
neurons that migrate along both the longitudinal and dorsoventral axes
during development, and rat pontine neuronal explants are unable to
respond to the netrin 1 guidance cue when mouse NAV1 is knocked
down by RNAi (Martinez-Lopez et al., 2005). Circumferential
guidance of growth cones in C. elegans is controlled by multiple
guidance cues, including UNC-6/netrin, which is expressed ventrally,
where it attracts UNC-40/DCC-expressing growth cones and repels
those expressing both UNC-40 and UNC-5 (Wadsworth, 2002).
Interestingly, UNC-34/Ena, which controls multiple aspects of cell
migration and guidance (Gitai et al., 2003; Shakir et al., 2006; Withee
et al., 2004; Yu et al., 2002), can suppress ectopic UNC-5 expression,
placing UNC-34 downstream of UNC-5 in circumferential guidance
(Colavita and Culotti, 1998). Mammalian Ena function is partially
dependent on ABI proteins, and could suggest a role for ABI-1 in
circumferential guidance in worms. (Comer et al., 1998; Juang and
Hoffmann, 1999; Tani et al., 2003).
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Development 136 (4)
Fig. 8. Excretory cell morphology in wild type, wve-1(rnai), nck-1(ok694) and arx-2(rnai) animals. (A-D) Fluorescence micrographs of
hermaphrodites carrying the ppgp-12::gfp transgene. The final positions of the posterior excretory canals are marked by arrows and the anus is
marked by arrowheads. Lateral views are shown in all cases except for B, which is a ventral view. Anterior is to the left. Scale bars: 100 μm. (A) Wildtype animal with posterior canals terminating near the anus. (B-D) RNAi-treated and mutant animals, as indicated have posterior excretory canals
that stop anterior to their wild-type positions (arrows). (E) Quantification of posterior longitudinal migration defects. Wild type (n=72), wsp1(gm324) (n=69), abl-1(ok171) (n=77), wve-1(rnai) (n=173), nck-1(ok694) (n=50), arx-2(rnai) (n=137), unc-53(n166) (n=55), wve-1(rnai); unc53(n166) (n=90), arx-2(rnai); unc-53(n166) (n=91).
WAVE activation (Innocenti et al., 2005). Our results indicate an
essential role for WAVE as opposed to WASP in longitudinal
outgrowth in C. elegans.
A model for UNC-53–ABI-1 action
Previously, it was shown that UNC-53 interacts physically with SEM5/GRB2 (Stringham et al., 2002), a SH2-SH3 adapter involved in
multiple RTK pathways, including FGFR (Dixon et al., 2006), EGFR
(Moghal and Sternberg, 2003), and IR (Hopper, 2006) signaling. At
present, it is unclear whether UNC-53 is a participant in one or several
signaling cascades. For example, whereas both unc-53 and egl15/FGFR are expressed in the migrating sex myoblasts (Goodman et
al., 2003), egl-15 is not expressed in axons (where it regulates
outgrowth) but instead exerts its effect through the underlying
hypodermis on which they migrate (Bulow et al., 2004). As UNC-53
is a cytoplasmic protein that functions cell autonomously, this suggests
that it does not act directly downstream of EGL-15/FGFR signaling
in neuronal cell migrations, but that it might be recruited by a different
receptor upstream of SEM-5/GRB2. Moreover, UNC-53, the celladhesion molecule UNC-71/ADAM and UNC-73/TRIO, have all
Fig. 9. Expression pattern of nck-1 using pnck1::gfp. (A) Adult hemaphrodite showing expression
in the excretory cell (Exc) and head neurons (HNeu).
(B) View of the midbody of adult hemaphrodite
showing expression in several motoneurons of the
ventral cord (VC). Scale bars: 50 μm.
DEVELOPMENT
defects observed in unc-53 and abi-1 animals are similar to those
observed in ced-10 and wve-1 (Lundquist et al., 2001; Lundquist,
2003; Withee et al., 2004), consistent with a model in which these
proteins operate together in cytoskeletal remodelling. ABI-1 is a
member of a complex consisting of WAVE-1 (WVE-1), GEX-2,
GEX-3 and HSPC300 that promotes actin nucleation (Innocenti et al.,
2004; Stroschein-Stevenson et al., 2006). The defects in cell
movements during morphogenesis reported for gex-2 and gex-3 (Soto
et al., 2002), and the similarities in the phenotypes of abi-1 and wve1 reported here are consistent with a model in which these proteins
form a similar complex in C. elegans.
Two models of ARP2/3 complex activation have been proposed;
one that relies on WAVE and another that relies on WASP (Bompard
and Caron, 2004; Stradal et al., 2004). ABI-1 seems to participate in
both, binding through its N terminus to WAVE to regulate membrane
protrusion and macropinocytosis, and through its SH3 domain to NWASP to stimulate actin-dependent vesicular transport and
endocytosis (Innocenti et al., 2005). Thus ABI-1 may be a central
figure that regulates the proportion of actin filament nucleation
designated for particular processes by partitioning WASP versus
RESEARCH ARTICLE
Table 1. Percentage of anteriorly directed PLM axons
truncated in eri-1(mg366); pmec-4::gfp
Genotype
wild type (n2)
unc-53(n166)
unc-53(rnai)
abi-1(rnai)
nck-1(rnai)
arx-2(rnai)
Percentage of truncated PLM axons
n
0.8
100
15
9.6
4
11
114
114
130
137
102
104
been implicated in a EGL-17/FGF-independent signaling mechanism
controlling sex myoblast migrations (Chen et al., 1997), suggesting
non-FGFR signaling is involved in this pathway as well. Therefore the
identity of ligands and receptors upstream of the SEM-5/UNC-53
interaction in cell migration remain elusive.
In this study, we found that a restricted region of the N terminus of
UNC-53 containing a CH domain was sufficient to bind ABI-1 in
vitro, and that the UNC-53–ABI-1 interaction mediated by this domain
is required for longitudinal cell outgrowth in vivo. CH domains are
commonly found in proteins involved in signal transduction and actin
binding, and are classified by the number and position of CH domains
they contain (Korenbaum and Rivero, 2002). Type 1/2 CH domain
proteins, such as α-actinin, β-spectrin and dystrophin, which function
in actin bundling and membrane anchoring (Broderick and Winder,
2005), possess two N-terminal CH domains in tandem. The first Type
1 CH domain mediates actin binding, whereas the second Type 2 CH
domain may (1) stabilize the actin interaction of the Type I domain, (2)
localize the actin-binding protein to the cytoskeleton, or (3) act as a
scaffold for signal transduction (Gimona et al., 2002). By contrast,
UNC-53 possesses a single N-terminal CH domain, and in this respect
is more closely related to Type 3 CH domain-containing proteins, such
as VAV, IQGAP, αPIX and SM22 (Gimona et al., 2002; Stradal et al.,
1998). Type 3 CH domains function like Type 2 CH domains in that
they act as scaffolds that bind proteins involved in the control of
cytoskeletal change and signal transduction (Galkin et al., 2006;
Gimona and Mital, 1998; Korenbaum and Rivero, 2002; Leinweber et
al., 1999). In such a model, UNC-53 may be a scaffold that coordinates
upstream signals transduced through SEM-5/GRB2 to ABI-1 and the
actin cytoskeleton.
The complexity of the unc-53 locus gives rise to several protein
isoforms that are regulated by different promoters and that display
non-overlapping tissue-specific expression patterns (Choi and
Newman, 2006; Stringham et al., 2002). The smaller isoforms are
under the control of intronic promoters, producing polypeptides that
lack CH domains, which might limit their ability to interact with ABI1 and significantly alter their function. Interestingly, both murine and
human NAV1 also lack CH domains (unlike mouse and human NAV2
and NAV3), and are the only NAV genes downregulated in brain
following development (Maes et al., 2002; Peeters et al., 2004),
suggesting a possible post-developmental role for the NAVs
possessing CH domains. Understanding the relationship between the
tissue specificity and the domain organization of the various isoforms
of UNC-53 and the vertebrate NAVs should shed light on the
importance of the CH domains in these proteins and how they operate.
We thank Laura Ramsay, Erin Kreiter, Will Bronec, Tanya Martens and
Samantha Grainger for technical assistance; the Caenorhabitis Genetics
Centre, Monica Driscoll, Shohei Mitani and Harald Hutter for nematode
strains; and Bob Barstead for the C. elegans yeast two-hybrid library. We are
grateful to Nancy Hawkins, Christopher Beh, and members of the Stringham
and Baillie labs for helpful discussions. J.W. was the recipient of an NSERC
USRA. This work was supported by NSERC Discovery Grants to E.G.S. and to
D.B., and by the Canada Research Chairs to E.G.S. and D.B.
573
References
Aspenstrom, P. and Olson, M. F. (1995). Yeast two-hybrid system to detect proteinprotein interactions with Rho GTPases. Methods Enzymol. 256, 228-241.
Bettinger, J. C., Lee, K. and Rougvie, A. E. (1996). Stage-specific accumulation of
the terminal differentiation factor LIN-29 during Caenorhabditis elegans
development. Development 122, 2517-2527.
Birnbaum, D., Popovici, C. and Roubin, R. (2005). A pair as a minimum: the two
fibroblast growth factors of the nematode Caenorhabditis elegans. Dev. Dyn. 232,
247-255.
Bompard, G. and Caron, E. (2004). Regulation of WASP/WAVE proteins: making a
long story short. J. Cell Biol. 166, 957-962.
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.
Broderick, M. J. and Winder, S. J. (2005). Spectrin, alpha-actinin, and dystrophin.
Adv. Protein Chem. 70, 203-246.
Bulow, H. E., Boulin, T. and Hobert, O. (2004). Differential functions of the C.
elegans FGF receptor in axon outgrowth and maintenance of axon position.
Neuron 42, 367-374.
Burdine, R. D., Chen, E. B., Kwok, S. F. and Stern, M. J. (1997). egl-17 encodes
an invertebrate fibroblast growth factor family member required specifically for sex
myoblast migration in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 94,
2433-2437.
Chen, E. B., Branda, C. S. and Stern, M. J. (1997). Genetic enhancers of sem-5
define components of the gonad-independent guidance mechanism controlling
sex myoblast migration in Caenorhabditis elegans hermaphrodites. Dev. Biol. 182,
88-100.
Choi, J. and Newman, A. P. (2006). A two-promoter system of gene expression in
C. elegans. Dev. Biol. 296, 537-544.
Colavita, A. and Culotti, J. G. (1998). Suppressors of ectopic UNC-5 growth cone
steering identify eight genes involved in axon guidance in Caenorhabditis elegans.
Dev. Biol. 194, 72-85.
Comer, A. R., Ahern-Djamali, S. M., Juang, J. L., Jackson, P. D. and Hoffmann,
F. M. (1998). Phosphorylation of Enabled by the Drosophila Abelson tyrosine
kinase regulates the in vivo function and protein-protein interactions of Enabled.
Mol. Cell. Biol. 18, 152-160.
Cordes, S., Frank, C. A. and Garriga, G. (2006). The C. elegans MELK ortholog
PIG-1 regulates cell size asymmetry and daughter cell fate in asymmetric
neuroblast divisions. Development 133, 2747-2756.
Courtney, K. D., Grove, M., Vandongen, H., Vandongen, A., LaMantia, A. S.
and Pendergast, A. M. (2000). Localization and phosphorylation of Ablinteractor proteins, abi-1 and abi-2, in the developing nervous system. Mol. Cell.
Neurosci. 16, 244-257.
Dai, Z. and Pendergast, A. M. (1995). Abi-2, a novel SH3-containing protein
interacts with the c-Abl tyrosine kinase and modulates c-Abl transforming activity.
Genes Dev. 9, 2569-2582.
DeVore, D. L., Horvitz, H. R. and Stern, M. J. (1995). An FGF receptor signaling
pathway is required for the normal cell migrations of the sex myoblasts in C.
elegans hermaphrodites. Cell 83, 611-620.
Dixon, S. J., Alexander, M., Fernandes, R., Ricker, N. and Roy, P. J. (2006). FGF
negatively regulates muscle membrane extension in Caenorhabditis elegans.
Development 133, 1263-1275.
Echarri, A., Lai, M. J., Robinson, M. R. and Pendergast, A. M. (2004). Abl
interactor 1 (abi-1) Wave-binding and SNARE domains regulate its
nucleocytoplasmic shuttling, lamellipodium localization, and Wave-1 levels. Mol.
Cell. Biol. 24, 4979-4993.
Eden, S., Rohatgi, R., Podtelejnikov, A. V., Mann, M. and Kirschner, M. W.
(2002). Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and
Nck. Nature 418, 790-793.
Frangioni, J. V. and Neel, B. G. (1993). Solubilization and purification of
enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal.
Biochem. 210, 179-187.
Funato, Y., Terabayashi, T., Suenaga, N., Seiki, M., Takenawa, T. and Miki, H.
(2004). IRSp53/Eps8 complex is important for positive regulation of Rac and cancer
cell motility/invasiveness. Cancer Res. 64, 5237-5244.
Galkin, V. E., Orlova, A., Fattoum, A., Walsh, M. P. and Egelman, E. H. (2006).
The CH-domain of calponin does not determine the modes of calponin binding to
F-actin. J. Mol. Biol. 359, 478-485.
Ghenea, S., Boudreau, J. R., Lague, N. P. and Chin-Sang, I. D. (2005). The VAB-1
Eph receptor tyrosine kinase and SAX-3/Robo neuronal receptors function
together during C. elegans embryonic morphogenesis. Development 132, 36793690.
Gimona, M. and Mital, R. (1998). The single CH domain of calponin is neither
sufficient nor necessary for F-actin binding. J. Cell. Sci. 111, 1813-1821.
Gimona, M., Djinovic-Carugo, K., Kranewitter, W. J. and Winder, S. J. (2002).
Functional plasticity of CH domains. FEBS Lett. 513, 98-106.
Gitai, Z., Yu, T. W., Lundquist, E. A., Tessier-Lavigne, M. and Bargmann, C. I.
(2003). The Netrin receptor UNC-40/DCC stimulates axon attraction and
outgrowth through Enabled and, in parallel, Rac and UNC-115/AbLIM. Neuron 37,
53-65.
DEVELOPMENT
UNC-53/NAV-2 interacts with ABI-1
RESEARCH ARTICLE
Goodman, S. J., Branda, C. S., Robinson, M. K., Burdine, R. D. and Stern, M. J.
(2003). Alternative splicing affecting a novel domain in the C. elegans EGL-15 FGF
receptor confers functional specificity. Development 130, 3757-3766.
Grove, M., Demyanenko, G., Echarri, A., Zipfel, P. A., Quiroz, M. E., Rodriguiz,
R. M., Playford, M., Martensen, S. A., Robinson, M. R., Wetsel, W. C. et al.
(2004). ABI2-deficient mice exhibit defective cell migration, aberrant dendritic
spine morphogenesis, and deficits in learning and memory. Mol. Cell. Biol. 24,
10905-10922.
Hao, J. C., Yu, T. W., Fujisawa, K., Culotti, J. G., Gengyo-Ando, K., Mitani, S.,
Moulder, G., Barstead, R., Tessier-Lavigne, M. and Bargmann, C. I. (2001). C.
elegans Slit acts in midline, dorsal-ventral, and anterior-posterior guidance via the
SAX-3/Robo receptor. Neuron 32, 25-38.
Hedgecock, E. M., Culotti, J. G. and Hall, D. H. (1990). The unc-5, unc-6, and
unc-40 genes guide circumferential migrations of pioneer axons and mesodermal
cells on the epidermis in C. elegans. Neuron 4, 61-85.
Hekimi, S. and Kershaw, D. (1993). Axonal guidance defects in a Caenorhabditis
elegans mutant reveal cell-extrinsic determinants of neuronal morphology. J.
Neurosci. 13, 4254-4271.
Higgs, H. N. and Pollard, T. D. (2000). Activation by Cdc42 and PIP(2) of WiskottAldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex.
J. Cell Biol. 150, 1311-1320.
Hopper, N. A. (2006). The adaptor protein Soc-1/Gab1 modifies growth factor
receptor output in Caenorhabditis elegans. Genetics 173, 163-175.
Ibarra, N., Pollitt, A. and Insall, R. H. (2005). Regulation of actin assembly by
SCAR/WAVE proteins. Biochem. Soc. Trans. 33, 1243-1246.
Innocenti, M., Tenca, P., Frittoli, E., Faretta, M., Tocchetti, A., Di Fiore, P. P. and
Scita, G. (2002). Mechanisms through which Sos-1 coordinates the activation of
Ras and Rac. J. Cell Biol. 156, 125-136.
Innocenti, M., Zucconi, A., Disanza, A., Frittoli, E., Areces, L. B., Steffen, A.,
Stradal, T. E., Di Fiore, P. P., Carlier, M. F. and Scita, G. (2004). Abi-1 is essential
for the formation and activation of a WAVE2 signaling complex. Nat. Cell Biol. 6,
319-327.
Innocenti, M., Gerboth, S., Rottner, K., Lai, F. P., Hertzog, M., Stradal, T. E.,
Frittoli, E., Didry, D., Polo, S., Disanza, A. et al. (2005). Abi-1 regulates the
activity of N-WASP and WAVE in distinct actin-based processes. Nat. Cell Biol. 7,
969-976.
Ishii, N., Wadsworth, W. G., Stern, B. D., Culotti, J. G. and Hedgecock, E. M.
(1992). UNC-6, a laminin-related protein, guides cell and pioneer axon migrations
in C. elegans. Neuron 9, 873-881.
Jenei, V., Andersson, T., Jakus, J. and Dib, K. (2005). E3B1, a human homologue
of the mouse gene product abi-1, sensitizes activation of Rap1 in response to
epidermal growth factor. Exp. Cell Res. 310, 463-473.
Juang, J. L. and Hoffmann, F. M. (1999). Drosophila Abelson interacting protein
(dAbi) is a positive regulator of Abelson tyrosine kinase activity. Oncogene 18,
5138-5147.
Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G. and Ahringer,
J. (2001). Effectiveness of specific RNA-mediated interference through ingested
double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2,
RESEARCH0002.
Kennedy, S., Wang, D. and Ruvkun, G. (2004). A conserved siRNA-degrading
RNase negatively regulates RNA interference in C. elegans. Nature 427, 645-649.
Kishi, M., Kummer, T. T., Eglen, S. J. and Sanes, J. R. (2005). LL5beta: a regulator
of postsynaptic differentiation identified in a screen for synaptically enriched
transcripts at the neuromuscular junction. J. Cell Biol. 169, 355-366.
Korenbaum, E. and Rivero, F. (2002). Calponin homology domains at a glance. J.
Cell. Sci. 115, 3543-3545.
Leinweber, B. D., Leavis, P. C., Grabarek, Z., Wang, C. L. and Morgan, K. G.
(1999). Extracellular regulated kinase (ERK) interaction with actin and the calponin
homology (CH) domain of actin-binding proteins. Biochem. J. 344, 117-123.
Levy-Strumpf, N. and Culotti, J. G. (2007). VAB-8, UNC-73 and MIG-2 regulate
axon polarity and cell migration functions of UNC-40 in C. elegans. Nat. Neurosci.
10, 161-168.
Lundquist, E. A. (2003). Rac proteins and the control of axon development. Curr.
Opin. Neurobiol. 13, 384-390.
Lundquist, E. A., Reddien, P. W., Hartwieg, E., Horvitz, H. R. and Bargmann, C.
I. (2001). Three C. elegans Rac proteins and several alternative Rac regulators
control axon guidance, cell migration and apoptotic cell phagocytosis.
Development 128, 4475-4488.
Maes, T., Barcelo, A. and Buesa, C. (2002). Neuron navigator: a human gene
family with homology to unc-53, a cell guidance gene from Caenorhabditis
elegans. Genomics 80, 21-30.
Martinez-Lopez, M. J., Alcantara, S., Mascaro, C., Perez-Branguli, F., RuizLozano, P., Maes, T., Soriano, E. and Buesa, C. (2005). Mouse Neuron
Navigator 1, a novel microtubule-associated protein involved in neuronal
migration. Mol. Cell. Neurosci. 28, 599-612.
McKay, S. J., Johnsen, R., Khattra, J., Asano, J., Baillie, D. L., Chan, S., Dube,
N., Fang, L., Goszczynski, B., Ha, E. et al. (2003). Gene expression profiling of
cells, tissues, and developmental stages of the nematode C. elegans. Cold Spring
Harb. Symp. Quant. Biol. 68, 159-169.
Development 136 (4)
Merrill, R. A., Plum, L. A., Kaiser, M. E. and Clagett-Dame, M. (2002). A
mammalian homolog of unc-53 is regulated by all-trans retinoic acid in
neuroblastoma cells and embryos. Proc. Natl. Acad. Sci. USA 99, 3422-3427.
Moghal, N. and Sternberg, P. W. (2003). The epidermal growth factor system in
Caenorhabditis elegans. Exp. Cell Res. 284, 150-159.
Nelson, F. K., Albert, P. S. and Riddle, D. L. (1983). Fine structure of the
Caenorhabditis elegans secretory-excretory system. J. Ultrastruct. Res. 82, 156171.
Pan, C. L., Howell, J. E., Clark, S. G., Hilliard, M., Cordes, S., Bargmann, C. I.
and Garriga, G. (2006). Multiple Wnts and Frizzled receptors regulate anteriorly
directed cell and growth cone migrations in Caenorhabditis elegans. Dev. Cell. 10,
367-377.
Peeters, P. J., Baker, A., Goris, I., Daneels, G., Verhasselt, P., Luyten, W. H.,
Geysen, J. J., Kass, S. U. and Moechars, D. W. (2004). Sensory deficits in mice
hypomorphic for a mammalian homologue of unc-53. Brain Res. Dev. Brain Res.
150, 89-101.
Sawa, M. and Takenawa, T. (2006). Caenorhabditis elegans WASP-interacting
protein homologue WIP-1 is involved in morphogenesis through maintenance of
WSP-1 protein levels. Biochem. Biophys. Res. Commun. 340, 709-717.
Sawa, M., Suetsugu, S., Sugimoto, A., Miki, H., Yamamoto, M. and
Takenawa, T. (2003). Essential role of the C. elegans Arp2/3 complex in cell
migration during ventral enclosure. J. Cell. Sci. 116, 1505-1518.
Shakir, M. A., Gill, J. S. and Lundquist, E. A. (2006). Interactions of UNC-34
Enabled with Rac GTPases and the NIK kinase MIG-15 in Caenorhabditis elegans
axon pathfinding and neuronal migration. Genetics 172, 893-913.
Shi, Y., Alin, K. and Goff, S. P. (1995). Abl-interactor-1, a novel SH3 protein binding
to the carboxy-terminal portion of the Abl protein, suppresses v-Abl transforming
activity. Genes Dev. 9, 2583-2597.
So, C. W., So, C. K., Cheung, N., Chew, S. L., Sham, M. H. and Chan, L. C.
(2000). The interaction between EEN and abi-1, two MLL fusion partners, and
Synaptojanin and Dynamin: implications for leukaemogenesis. Leukemia 14, 594601.
Soto, M. C., Qadota, H., Kasuya, K., Inoue, M., Tsuboi, D., Mello, C. C. and
Kaibuchi, K. (2002). The GEX-2 and GEX-3 proteins are required for tissue
morphogenesis and cell migrations in C. elegans. Genes Dev. 16, 620-632.
Stradal, T., Kranewitter, W., Winder, S. J. and Gimona, M. (1998). CH domains
revisited. FEBS Lett. 431, 134-137.
Stradal, T., Courtney, K. D., Rottner, K., Hahne, P., Small, J. V. and Pendergast,
A. M. (2001). The Abl interactor proteins localize to sites of actin polymerization at
the tips of lamellipodia and filopodia. Curr. Biol. 11, 891-895.
Stradal, T. E., Rottner, K., Disanza, A., Confalonieri, S., Innocenti, M. and Scita,
G. (2004). Regulation of actin dynamics by WASP and WAVE family proteins.
Trends Cell Biol. 14, 303-311.
Stringham, E. G., Dixon, D. K., Jones, D. and Candido, E. P. (1992). Temporal and
spatial expression patterns of the small heat shock (hsp16) genes in transgenic
Caenorhabditis elegans. Mol. Biol. Cell 3, 221-233.
Stringham, E., Pujol, N., Vandekerckhove, J. and Bogaert, T. (2002). unc-53
controls longitudinal migration in C. elegans. Development 129, 3367-3379.
Stroschein-Stevenson, S. L., Foley, E., O’Farrell, P. H. and Johnson, A. D. (2006).
Identification of Drosophila gene products required for phagocytosis of Candida
Albicans. PLoS Biol. 4, e4.
Takenawa, T. and Suetsugu, S. (2007). The WASP-WAVE protein network:
connecting the membrane to the cytoskeleton. Nat. Rev. Mol. Cell. Biol. 8, 37-48.
Tani, K., Sato, S., Sukezane, T., Kojima, H., Hirose, H., Hanafusa, H. and
Shishido, T. (2003). Abl interactor 1 promotes tyrosine 296 phosphorylation of
mammalian Enabled (Mena) by c-Abl kinase. J. Biol. Chem. 278, 21685-21692.
Tanos, B. E. and Pendergast, A. M. (2007). Abi-1 forms an epidermal growth
factor-inducible complex with Cbl: role in receptor endocytosis. Cell. Signal. 19,
1602-1609.
Volkmann, N., Amann, K. J., Stoilova-McPhie, S., Egile, C., Winter, D. C.,
Hazelwood, L., Heuser, J. E., Li, R., Pollard, T. D. and Hanein, D. (2001).
Structure of Arp2/3 complex in its activated state and in actin filament branch
junctions. Science 293, 2456-2459.
Wadsworth, W. G. (2002). Moving around in a worm: Netrin UNC-6 and
circumferential axon guidance in C. elegans. Trends Neurosci. 25, 423-429.
Watari-Goshima, N., Ogura, K., Wolf, F. W., Goshima, Y. and Garriga, G.
(2007). C. elegans VAB-8 and UNC-73 regulate the SAX-3 receptor to direct cell
and growth-cone migrations. Nat. Neurosci. 10, 169-176.
Withee, J., Galligan, B., Hawkins, N. and Garriga, G. (2004). Caenorhabditis
elegans WASP and Ena/VASP proteins play compensatory roles in morphogenesis
and neuronal cell migration. Genetics 167, 1165-1176.
Yu, T. W., Hao, J. C., Lim, W., Tessier-Lavigne, M. and Bargmann, C. I. (2002).
Shared receptors in axon guidance: SAX-3/Robo signals via UNC-34/Enabled and a
Netrin-independent UNC-40/DCC function. Nat. Neurosci. 5, 1147-1154.
Zallen, J. A., Yi, B. A. and Bargmann, C. I. (1998). The conserved immunoglobulin
superfamily member SAX-3/Robo directs multiple aspects of axon guidance in C.
elegans. Cell 92, 217-227.
Zhao, Z., Fang, L., Chen, N., Johnsen, R. C., Stein, L. and Baillie, D. L. (2005).
Distinct regulatory elements mediate similar expression patterns in the excretory
cell of Caenorhabditis elegans. J. Biol. Chem. 280, 38787-38794.
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